Tools of the Laboratory

1. Tools of the Laboratory This lecture will address the types of media used for studying microbes in the lab as well as there basic nutrient needs.

2. All organisms require certain physical or environmental conditions to grow. If those physical conditions are not met, the organism’s growth will either be inhibited or it will die. • Temperature is an important physical condition. There are several different groups of organisms that prefer different temperature ranges for growth. Microbes that cause disease generally prefer a growing temperature equal to body temperature (37 degrees C). • Psychrophiles: A group of cold-loving microbes that generally grow at temperatures of -10 to 20 degrees C. • Mesophiles: A group of moderate-temperature-loving microbes that generally grow at temperatures of 10 to 50 degrees C. Pathogenic microorganisms are in this group. Refrigeration severely retards the growth of most pathogenic bacteria • Thermophiles: A group of heat-loving microbes that generally grow at temperatures of 40 to 73 degrees C.

3. pH is another important physical characteristic for growth. Remember that the pH scale is from 0-14. A low pH = acidic whereas a high pH = basic and 7 is neutral. • The normal growth range for bacteria is pH 6.5 to 7.5. Notice that the body’s pH is also within that range. (Most bacteria can’t grow in orange juice because it is too acidic. So why does orange juice spoil?) • Acidophiles are a small group of bacteria that can grow in pH 4 conditions. These organisms are non-pathogenic. • Molds and yeasts grow in pH 5-6. • Have you figured out the answer to the orange juice question? It is usually molds and yeasts that spoil the orange juice with their metabolic by products.

4. Osmotic pressure is used all the time in the food industry to for preservation. • It is the addition of large quantities of salts or sugars to foods that result in shrinkage of cells due to the loss of water. • Plasma membrane pulls away from the cell wall which results in inhibition of cell growth. • Ex. In food prep = salted fish, honey, sweetened and condensed milk, pickles • All of the items above are preserved because of a large amount of salt or sugar. • Halophiles: A group of organisms that can grow in high salt concentrations.

5. All organisms also need some basic nutrients to sustain growth. • Carbon: makes up proteins, lipids, and carbohydrates • N, S, P: makes amino acids, ATP, DNA, RNA • Trace elements: iron, copper, molybdenum, zinc • Used by enzymes for proper functioning. • Organic growth factors: needed for growth that cannot be synthesized by own enzymes (ie. Vitamins in humans) • Oxygen, ATP, water • If any of these major nutrients are missing then the organism cannot function properly and eventually stops growing (forms endospore) or dies.

6. How microbes feed • Heterotroph: A group of microbes that use organic carbon as their carbon source for metabolism. • Autotroph: A group of microbes that use inorganic carbon as their carbon source, such as carbon dioxide. • Phototroph: A group of microbes that use light as their energy source. • Chemotroph: A group of microbes that use chemical compounds for their energy source, such as glucose. • Photoautotroph • Chemoautroph • Photoheterotroph • Chemoheterotroph • Saprobe: A group of organisms that use decaying matter as it’s carbon and energy source. • Parasite: Requires a host to get it’s carbon and energy sources.

7. Gas Requirements • Aerobe: A group of organisms that use oxygen for metabolism. • Facultative Anaerobe: A group of organisms that do not require oxygen for metabolism. • Microaerophile: A group of organisms that require small amounts of oxygen for metabolism. • Strict Anaerobe: A group of organisms that require an element other than oxygen for metabolism, such as N, S, CO2.

8. Ecological Association Among Microorganisms • Symbiosis: 2 organisms live together in a close partnership. There are several different types of symbiotic relationships. • Mutualism: Both organisms benefit from the relationship. For example, E.coli found in our intestines aids in our digestion and it benefits from us because our intestines provide a nice, warm, nutrient rich environment for it to grow. • Commensalism: In this relationship one organism benefits and the other is unaffected. For example, microbes that feed off of the dead skin cells that we shed are benefited by us but we are unaffected by them. • Parasitism: In this relationship one organism benefits at the expense of the other. A tape worm is an example of this relationship. The tape worm attaches itself to the intestine of the host. The host provides a nice, warm, nutrient rich environment. The tape worm grows and grows. It can eventually cause malnutrition or intestinal blockages that are harmful to the host.

9. Synergism: This type of relationship is one in which there is an added effect by the close relationship. • An example is found in biofilms. Many of you found that many groups of organisms make up a biofilm and that when these organisms are in a close relationship like that, new genes are turned on that benefit the whole group. Those genes are not activated when the organisms are alone. • Antagonism: This type of relationship is a competitive relationship between organisms. • For example, many organisms make personal antibiotics that kill off competitors for nutrients. Many of the antibiotics that we use today were originally derived from these natural antibiotics. Penicillin is one!

10. Uses of Media • Because we understand the basic principles of nutrition for living cells, we can use that information to study them in the lab. • The media that we used in lab to grow microorganisms is designed according to the metabolic properties of microorganisms. • There are three basic properties that are used when deciding which media to use for growth of microbes. • 1. Physical state • Liquid:broth in a test tube. (The nutrient broth that we use in lab is an example of this type of media.) • Semisolid: broth that has a little agar added to it so that it has a thicker consistency but is not solid. (The motility agar that we used in our last lab is an example of this type of media.) • Solid: broth that has enough agar added to it to make it solid. (The agar plates that we have been using in lab are examples of this type of media.)

11. 2. Chemical composition: We can manipulate the media to select for the growth of one organism and inhibit the growth of another. We can also manipulate the media to show us metabolic differences between organisms. • Complex media is the type of media we have used thus far in lab. (Remember we talked about chicken broth for microorganisms. It is derived from yeast, meat, or plant digests and contains all the nutrients that a microbe needs to grow. In this type of media the amount of nutrients varies from batch to batch and is unknown. • Chemically defined media is type of media in which the exact chemical or nutrient composition is known. Each nutrient is carefully weighed and added one by one to the media. In order for this media to support bacterial growth an organism’s nutrient requirements must be known and planned for.

12. 3. Functional type: This category of media determines if it will select for the growth of one type of organism or if it will show metabolic differences between organisms. • Selective media: This type of media suppresses the growth of unwanted bacteria and encourages the growth of desired organism. • For example, let’s say that we have a test tube that contains both G+ and G- bacteria in it. You want to study the G+. To help you isolate the G+ from the G- bacteria you can use a media that inhibits the growth of G- bacteria, leaving only the G+. • Differential Media • Differential media makes it easy to distinguish different genus and species of organisms. • Blood agar is an easy way to distinguish between some organisms. It is media that contains sheep blood. It is red and solid. You can’t see through it because of the red blood cell content. Some organisms have the ability to break down the red blood cells, S. pyogenes. When the red blood cells are broken down the media turns orange and transparent. Some organisms can’t break down the red blood cells so the media appears unchanged.

13. Enrichment Media • Allows for growth of microbes that are not normally detected. Some microbes grow in very small numbers because their nutrient requirements are not optimal. This type of media allows you to add the specific nutrient required to select for that one type of bacteria and encourage it’s growth so that it can be isolated from the rest. • For example, you take a soil sample and want to study the organisms found within the sample. One of the organisms requires phenol as carbon and energy source. That is an unusual growth requirement so it has to be added to encourage the growth of that particular organism. When it is added to the media it will select for the growth of the organism you want, while other organisms are unable to grow in the presence of phenol.

14. Some special culture techniques are used to culture hard to grow organisms • Ie. Mycobacterium, the organism that is responsible for Leprosy and Tuberculosis, has a low temperature requirement for growth. (Slightly lower than body temperature.) To study it in the lab it is grown in armadillos or in the foot pads of mice and rats because the body temperature is ideal. • Some organisms only grow inside cells, such as viruses. So special tissue cultures have to be grown in order to replicate the viruses. • Some organisms still cannot be grown in the lab because their growth requirements are not understood well enough.

15. Growth of Bacterial Cultures • Bacterial Division: Bacteria reproduce through binary fission. This produces 2 identical cells each time the parent cell divides. See Fig.7.13 for a great explanation on this concept. • The Generation Time of a microbe is the time required for a cell to divide and the population to double. • For example, E-coli doubles once every 20 min. 10 cells become 20 cells after 20 minutes and 20 cells become 40 cells after 20 additional minuets, etc. • The following is the equation used to calculate the final population of a bacterial culture: nf=ni(2)g nf is the final populationni is the initialpopulation g is the number of generations 2 represents the doubling due to binary fission • Try calculating the following problem. (I’ll go over it in lab on Friday.) • A culture was inoculated with 10 E-coli cells. Then it was put in the incubator for 2 hours. What was the final population? • Hint: first calculate the number of generations by figuring out how many times E.coli can divide within 2 hours if it has a doubling time of 20 minutes.

16. Bacterial Growth Curve • 4 basic phases of growth: • 1. Lag phase: New growth medium is added or a new culture is made. This period is a time of delayed growth while the bacterial cells prepare to divide. DNA is replicating, the cell wall and membrane or expanding, etc. • 2. Log phase (exponential growth phase): Cellular division is most active during this period. The generation time reaches a constant minimum. • 3. Stationary phase: A state of equilibrium where the number of cell deaths equals the number of cell divisions. • 4. Death phase: The number of cell deaths exceeds the number of new cells. The nutrients are becoming depleted or a change in the physical conditions are making conditions unfavorable. • Early death phase is when sporulation begins. • See Fig.7.15 for a great example of the shape of the growth curve.

17. Preserving bacterial cultures Once we are able to grow our bacteria we want to be able to preserve them so we don’t have to go through the same tedious process to isolate and grow them. A couple of the methods that are used are • 1. Deep-freezing: liquid bacterial culture is added to liquid glycerin to prevent breaking of membrane due to freezing. Then it is frozen at temp. ranging from –50 to –72 degrees C. • Lyophiliztion (freeze drying): The bacterial culture is quickly frozen at temp. ranging from –54 to – 72 degrees C. Then the water is removed by a vacuum leaving a powder like residue. The powder contains the organisms. • I’ll post the homework for this lecture on Thursday morning. 9/14/06 It will be due on Monday.